Arsenic exposure at high doses is neurotoxic to the mature nervous system. However, relatively little is known about the effects of arsenic exposure on the developing nervous system, which is often more sensitive to toxicological insults than the mature nervous system. Arsenic contamination of groundwater exposes millions of people in the US and worldwide to elevated levels of arsenic, and there is increasing evidence that early exposure to arsenic results in reduced mental function in children, although the cellular and molecular mechanisms for this are unknown. Our preliminary data, using an in vitro system, show that exposure of neurons during differentiation to low micromolar arsenic results in "stunted" neurons with reduced growth and complexity. We also find associated abnormal distribution and organization of microtubules and actin, which are key regulators of neurite growth, in exposed neurons. Furthermore, we find that arsenic induced reductions in neurite growth can be partially rescued by inhibiting the RhoA pathway. Arsenic disruption of microtubules and actin in non-neuronal cell types has been documented, although the molecular mechanisms are not well understood. Our long term objective is to understand the molecular mechanisms by which arsenic exposure during development impairs neurite growth, and the consequences this has for the structure and function of the nervous system. The goal of this grant is to focus on two regulators of cytoskeletal function;(i) Rho GTPases which are major regulators of actin dynamics in developing neurons and (ii) microtubule associated proteins (MAPs) which regulate microtubule stability. We test the hypothesis that arsenic disruption of Rho GTPases and MAPs drives disrupted actin and tubulin regulation which results in reduced neurite growth and branching. The first specific aim is to determine the extent to which arsenic induced disruption of Rho GTPase activity drives altered actin function and contributes to reduced neurite growth and complexity. Arsenic effects on Rho GTPase activity and levels will be quantified. Mutant Rho GTPases will then be used to modulate Rho GTPase activity in arsenic exposed neurons to determine the extent of their contribution. The second specific aim is to test the hypothesis that arsenic disruption of MAPs alters associated microtubule stability and polymerization. Arsenic effects on MAP production, phosphorylation and function will be investigated and quantified. Associated effects on microtubule stability and polymerization will be investigated and used to guide experiments where microtubule dynamics will be manipulated in arsenic exposed neurons to further elucidate their role in the effects of arsenic on neuron growth. PUBLIC HEALTH RELEVANCE Chronic low-dose arsenic exposure is a problem in regions of the US where drinking water is contaminated with arsenic. Surprisingly little is known about the consequences of this exposure for the developing nervous system, despite the potential for significant numbers of children and fetuses to be exposed to arsenic via contaminated water. This research will investigate how arsenic exposure disrupts the growth of the neurons which make up the nervous system.